AREA 1. MEMBRANE PROTEINS IN BIOENERGETICS The research focus in Area 1 has been the ATP synthase. This enzyme produces 90% of cellular ATP through a unique rotary mechanism driven by the transmembrane flow of H+ or Na+ ions. Many of the key aspects of this important mechanism remain poorly understood. In the FY14/15 period, our investigations have focused on establishing the structural principle that explains the ion specificity of the enzyme, which varies across different species. In particular we sought to experimentally test a theory of ion selectivity we had previously proposed (Refs. 1-3) and to further elaborate on that theoretical principle. Specifically, we set out to measure the Na+/H+ selecitivity of an isolated c-ring specifically that from the Na+-dependent ATP synthase of I. tartaricus, which our previous computational analyses had shown to be 1000-fold H+ selective (Refs. 1-3). To this end, we established and carried out a novel Isothermal Calorimetry (ITC) assay, with protein samples and equipment kindly provided by my collaborator Dr. Thomas Meier (Max Planck Institute of Biophysics). Specifically, we determined the apparent Na+ affinity of the c-ring as a function of pH, in conditions in which the ITC signals reflect the competition between Na+ and H+. From this data, we quantified the absolute Kd for both ions, and thus the intrinsic selectivity of the c-ring. Measured and theoretical values were found to be in excellent quantitative agreement, thus validating the principle of selectivity we previously derived computationally. The Kd values for the isolated c-ring were also found to be in good agreement with Km values found in the literature validating the notion that the specificity of the enzyme is conferred by the thermodynamic selectivity of the c-ring. In parallel with this experimental work, we expanded our theoretical analysis to more specifically analyze how subtle variations in the structural dynamics of the ion-binding sites, for an identical or highly similar chemical make-up (which is the primary factor) contributes to fine-tune the binding specificity of these sites. This study was published in the Proceedings of the National Academy of Sciences (see Bibliography, Leone et al). AREA 2. TRANSPORT AND SIGNALING ACROSS BIOLOGICAL MEMBRANES Two classes of membrane transporters of biomedical significance have been the focus in Area 2. First, RND multidrug-efflux pumps, which confer antibiotic resistance to pathogenic bacteria. Second, the CaCA superfamily of cation/Ca2+ exchangers, which are key for the homeostasis of cellular calcium, e.g. during the heartbeat. Specifically, we sought to use computational methods to delineate the mechanism of the RND-type homotrimeric proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. This study was carried in collaboration with Prof. Martin Pos (University of Frankfurt), who provided high-resolution X-ray crystallography data. Analyses of wildtype AcrB and four functionally-inactive variants revealed an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 apart. Each of these cycles entail two distinct types of collective motions of two structural repeats, coupled by flanking &#945;-helices that project from the membrane. In addition, we rationalized how the interactions among protomers across the trimerization interface might lead to a more kinetically efficient efflux system. This study was reported in the journal eLife (see Bibliography, Seeger et al). In the area of CaCA-family transporters, we sought to identify the functional state of the only available atomic structure of a Na+/Ca2+ exchanger, namely that of NCX_Mj from Methanococcus jannaschii, a homolog of cardiac NCX. This study was carried out in collaboration with Prof. Daniel Khananshvili (University of Tel Aviv), who provided biochemical assays. Specifically, we used molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. We found that in this most probable configuration, three Na+ ions occupy the so-called S(ext), S(Ca), and S(int) sites, whereas the S(mid) site is occupied by one water molecule and one H+, which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na+/Ca2+ and Ca2+/Ca2+ exchange by wild-type and mutagenized NCX_Mj confirmed that transport of both Na+ and Ca2+ requires protonation of D240, and that this side chain does not coordinate either ion at S(mid). These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na+ across the membrane is electrogenic, whereas transport of Ca2+ is not. Altogether, these findings provided the basis for ongoing experimental and computational studies of the conformational mechanism of this exchanger. This study was published in the Proceedings of the National Academy of Sciences (see Bibliography, Marinelli et al). AREA 3. DEVELOPMENT OF SIMULATION METHODS Our efforts in Area 3 have been focused on advancing the development and validation of a novel simulation methodology to identify and characterize minimum free-energy (i.e. most probable) pathways in complex conformational transitions and chemical reactions in biomolecules. We refer to this approach as String Method with Optimal Molecular Alignment, or SOMA (Ref. 4). In the FY14/15 period, we applied this powerful methodology to characterize the atomic mechanism underlying the interconversion between different isoforms of ATP-Mg2+ in solution a problem that required an analysis of the system in a 48-dimensional space. The results were successfully contrasted with previous theoretical and experimental work, including NMR measurements. This study was published in the Journal of Computational Chemistry (see Bibliography: Branduardi et al). In parallel to our work on SOMA, we introduced a novel molecular simulation method we refer to as Ensemble-Biased Metadynamics (EBMetaD). This method biases a conventional molecular dynamics simulation so as to sample a conformational ensemble that is exactly consistent with one or more probability distributions known a priori, e.g., experimental intramolecular distance distributions obtained by double electron-electron resonance or other spectroscopic techniques. Unlike conventional approaches used in structural refinement, the bias introduced in EBMetaD is the minimum necessary to fulfill the target distributions, i.e., EBMetaD satisfies the maximum-entropy principle. Among the existing methods in this class, EBMetaD is to our knowledge the most computationally efficient and straightforward in practice. This work was published in Biophysical Journal (see Bibliography, Marinelli & Faraldo-Gomez). REFERENCES 1. Krah A ... Faraldo-Gomez JD. Structural and energetic basis for H+ versus Na+ binding selectivity in ATP synthase Fo rotors. Biochim Biophys Acta 1797, 763-772 (2010) 2. Mayer F, Leone V ... Faraldo-Gomez JD, Mller V. A c-subunit with four transmembrane helices and one ion (Na+)-binding site in an archaeal ATP synthase: implications for c ring function and structure. J Biol Chem 287, 39327-39337 (2012) 3. Schlegel K, Leone V, Faraldo-Gomez JD, Mller V. Promiscuous archaeal ATP synthase concurrently coupled to Na+ and H+ translocation. Proc Natl Acad Sci USA 109, 947-952 (2012) 4. Branduardi D, Faraldo-Gomez JD. String method for calculation of minimum free-energy paths in Cartesian space in freely-tumbling systems. J Chem Theory Comput 9, 4140-4154 (2013)